Conditions: F buffer containing 1 mM ADP and 40 nM cofilin. Severing of actin filaments occurred within a cofilin cluster (at 53 s) and then at or near the boundary between a bare zone and another cofilin cluster (at 64.5 s). Imaged at 2 frames/s and played at 5 frames/s. Red, blue, and green arrowheads indicate severing in half helices in cofilin clusters, in a bare half helix immediately neighboring a cofilin cluster, and in bare zones more than half a helix away from cofilin clusters, respectively. White arrowheads: cofilin clusters. Z-scale was 0–12 nm. Related to Figure 6A.

( A – C ) are electron micrographs of negatively stained samples, and ( D ) is a HS-AFM image of a sample similar to that shown in ( B ). ( A ) Actin filaments bound with cofilin-rod. Arrowheads show crossover points in clusters of cofilin-rod. The rod portions of the fusion proteins are not readily visible, which may be due to alignment of the rods along the cofilin clusters. Severing activity and stoichiometric binding of cofilin-rod to actin filaments were confirmed by HS-AFM (Video 1) and co-sedimentation assays (Figure 3—figure supplement 3), respectively. ( B ) Cofilin-rod molecules sparsely bound to actin filaments, identified by the rod-like structures (black arrowheads). ( C ) Actin filaments with bound cofilin molecules (without rod fusion). Arrowheads show crossover points in clusters. Actin filaments and cofilin or cofilin-rod were mixed at a 2:1 ( A and C ) or 1:1 ( B ) molar ratio in F buffer containing 1 mM ATP. Bars: 25 nm. ( D ) HS-AFM image of an actin filament and a

The first three quarter of this video were taken in F buffer containing 1 mM ATP and 1 µM cofilin, and the last one quarter was taken in the presence of 650 nM cofilin. In the presence of 1 µM cofilin, filaments were fully decorated along the length, and no severing was observed. In the presence of 650 nM cofilin, there were some bare zones, and severing occurred near the boundary of the bare zone and the cofilin clusters, regardless of the size of cofilin clusters. For color codes of the arrowheads, see the legend to Video 6. Images were taken at 2 frames/s and played at 5 frames/s. Z-scale was 0–12 nm. Related to Figure 6—figure supplement 4.

In this video, seven different image sequences from different filaments and experiments were merged. Conditions: F buffer containing 1 mM ATP and 75 nM cofilin, except in sequence 3 in which cofilin concentration was 150 nM. For color codes of the arrowheads, see the legend to Video 6. Z-scale was 0–12 nm. Related to Figure 6—figure supplement 2.

( A and B ) HS-AFM image of a short cofilin cluster transiently associating with two S1 molecules (yellow arrowheads), which persisted for ∼1 s, enabling identification of the filament polarity ( B ). Measurements were made in F buffer containing 20 nM S1, 75 nM cofilin, 1 mM ADP, and 0.1 mM ATP. Bar: 25 nm; Z-scale: 0–12 nm. ( C and D ) Time-dependent changes in the heights of the indicated peaks (white arrowheads) and half helical pitches between the indicated peaks. ( E and F ) Histograms of the lengths of the half helical pitches of bare actin segments immediately neighboring cofilin clusters. Filaments were incubated in F buffer containing 1 mM ADP and 0.1 mM ATP for 5 min before observation ( E ) as in ( A and B ) or were incubated in F buffer containing 1 mM ADP for 30 min before observation ( F ). Pitches of the half helices of the first immediate neighbor on each side of cofilin clusters were measured. These values, together with those for the second neighbors, are summarized

The growth of cofilin clusters was observed under three buffer conditions: in the presence of 1 mM ADP and 0.1 mM ATP (+ADP +ATP); 1 mM ADP (+ADP) and 1 mM ADP and 10 mM Pi (+ADP +Pi), as in Figure 4. Growth of a cluster by one-half helix was counted as one growth event. The total number of observed growth events was 37, 46, and 188 for each condition. We speculate that at least some of the cluster growth events in the barbed-end direction were actually growth in the preferred direction from invisibly small clusters on the barbed side of a visible cluster.

( A and B ) show predicted peak heights of control actin filaments (PDB ID 3G37; Murakami et al. (2010)), and ( C and D ) show fully cofilin-decorated filaments (PDB ID 3J0S; Galkin et al. (2011)). These images were generated using Chimera software. When the true crossover point coincides with the position of an actin subunit ( A ) or a bound cofilin molecule ( C ), the position of the peak is equal to that of the crossover point, and its height is the tallest of the various possible orientations of the filament on the substrate. When the true crossover point is at the middle of two actin subunits ( B ) or two bound cofilin molecules ( D ), the peak height is the shortest of the possible orientations. In this latter case, the observed peak position is away from the true crossover point by as much as ∼2.5 nm. Depending on the orientation of the filament, therefore, the distance between the observed peak position and the true crossover point fluctuates between 0 and 2.5 nm. Random distr

( A and B ) are SDS-PAGE of supernatant and pellet fractions after ultracentrifugation of mixtures of various concentrations of cofilin with ( A ) or without ( B ) His-tag and 1 µM actin filaments, respectively. Acrylamide concentration of the gel was 12%. His-tag was removed by incubation with 1/4 (wt/wt) of His-tagged TEV protease overnight at 5°C and then passed through a Ni-column in the presence of 10 mM imidazole. Lanes 1 to 4 in ( C ) show His-TEV protease, mixture of His-cofilin and His-TEV after reaction over night, His-cofilin, and the reaction mixture that flowed through a Ni-NTA column (i.e., cofilin without His-tag). Co-sedimentation experiments were performed as follows. Frozen aliquots of cofilin (±His-tag) and rabbit skeletal actin were thawed on ice for 1 hr and were then clarified by ultracentrifugation at 80,000 rpm for 5 min at 5°C. The supernatant was collected, and protein concentrations were measured using Advanced Protein Assay reagent (Cytoskeleton). Actin fil

( A and B ) SDS-PAGE analysis of binding of cofilin-rod with and without His-tag to actin filaments, respectively. Experiments were carried out in F buffer containing 1 mM ATP, and protein concentrations used are shown above each gel. Cleavage of His-tag by TEV protease was performed as described in the legend to Figure 3—figure supplement 1. Acrylamide concentration of the gel was 10%.

Conditions: F buffer containing 1 mM ATP and 75 nM cofilin-rod without His-tag, imaging rate: 4 frames/s, and playing rate: 5 frames/s. Transient binding of cofilin-rod to and dissociation from an actin filament was followed for approximately 90.5 s, shown by the presence and absence of a blue arrowhead. No severing was observed in all four similar cases of successful imaging of sparse binding of cofilin-rod to actin filaments. Z-scale was 0–12 nm. Related to Figure 3D.

Imaged at 2 frames/s and played at 5 frames/s. White arrowheads show growth of the cofilin cluster, and yellow and magenta arrowheads show binding of S1. Magenta arrowheads indicate S1 molecules whose binding angle could not be determined, either for geometric reasons (i.e., binding on the upper face of the filament) or because the binding was too short-lived. Z-scale was 0–12 nm. For magnifications and polarity of the analyzed filaments, refer to Figure 4 in the main text. Related to Figure 4A.

Imaged at 2 frames/s and played at 5 frames/s. For color codes of arrowheads, see the legend to Video 3. Z-scale was 0–12 nm. For magnifications and polarity of the analyzed filaments, refer to Figure 4 in the main text. Related to Figure 4B.

For color codes of arrowheads, see the legend to Video 3. For magnifications and polarity of the analyzed filaments, refer to Figure 4 in the main text. Under this condition, binding of S1 was so short-lived that the tilted binding was not obvious in some cases (magenta). The S1 molecule indicated by a double magenta arrowhead appears to tilt in the direction opposite to other S1 molecules indicated by yellow arrowhead, and we speculate that this is because this S1 molecule was not stably bound to the filament when imaged. Imaged at 2 frames/s and played at 3 frames/s. Z-scale was 0–12 nm. Related to Figure 4C.

To show more general view of severing events, in addition to the small number of representative cases shown in Videos 6–8, 10 different image sequences from different experiments were merged. Sequence numbers are shown in the first 10 frames of each sequence. For color codes of the arrowheads, see the legend to Video 6. Conditions: F buffer containing 1 mM ATP (sequences 1–6), 1 mM ADP (sequence 7–8) or 1 mM ATP + 10 mM Pi (sequence 9–10). The concentration of cofilin shown in this video was 75 nM, except in sequences 9 and 10, in which it was 300 and 150 nM, respectively. Z-scale was 0–12 nm. Related to Figure 6—figure supplement 1.

Four independent image sequences are merged. Conditions: F buffer containing 1 mM ATP and 0, 10 or 40 nM cofilin without His-tag. In this video, three data sets which represent three cases of the absence or presence of cofilin are sequentially shown and indicated before each sequence begins as (i) Control: Without Cofilin, (ii) 10 nM Cofilin (two different image sequences), and (iii) 40 nM Cofilin. Note that severing of actin filaments was not observed not only in the absence but also in the presence of 10 nM cofilin. In the presence of 40 nM cofilin, severing was infrequently observed. For color codes of the arrowheads, see the legend to Video 6. Images were taken at 0.5 frames/s, except in the presence of 40 nM cofilin they were recorded at 0.25 frames/s, and the video is played at 5 frames/s. Z-scale was 0–12 nm. Related to Figure 6—figure supplement 3.